Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from chickens in a diagnostic laboratory

The aim of this study was to determine the resistance profile of 24 Campylobacter jejuni and 16 Campylobacter coli isolates from chickens in a diagnostic laboratory in Nigeria. Susceptibility testing was done by a broth microdilution MIC method with MICRONAUT – S anaerob test plates (Merlin Diagnostika, GmbH, Germany). MIC assay was performed according to CLSI (formally NCCLS) methods. Resistance to ciprofloxacin (57.5%) was the highest, followed by nalidixic acid (47%), tetracycline (35%) and trimethoprim/sulphamethoxazole (22%). Campylobacter jejuni were more resistant than Campylobacter coli to nalidixic acid, tetracycline and trimethoprim/sulphamethoxazole while Campylobacter coli were more resistant than Campylobacter jejuni to erythromycin and streptomycin. 47.5% of the isolates were multi drug resistant with nalidixic acid and ciprofloxacin as the most frequently occurring antimicrobial agent in the pattern. This work has shown that majority of the Campylobacter isolates were resistant to most of the antimicrobial agents used with multi drug resistance, thus the need for surveillance and rational use of antimicrobial agents in poultry production.


INTRODUCTION
Campylobacter species are among the leading cause of bacterial enteritis in humans throughout the world (Friedman et al., 2000).Campylobacteriosis is a zoonotic disease with domestic animals as well as wild animals acting as reservoirs for Campylobacter species (Padungton and Kaneene, 2003).The emergence of antimicrobial resistance species due to the use of antimicrobial agents in husbandry is a matter of concern (Luber et al., 2003).Several studies have linked the use of antimicrobial agents in Veterinary Medicine to the emergence and spread of resistance among Campylobacter with potentially serious effects on food safety in both veterinary and human health (Endtz et al., 1991;Deckert et al., 2010;Economou et al., 2015).*Corresponding author.E-mail: ssngun@yahoo.com.Tel: +2348036014337.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Campylobacter resistance to antimicrobial agents have been reported in both developed and developing countries (Cardinale et al., 2003;Ge et al., 2013;Nobile et al., 2013).The situation seems to be more in developing countries where there is wide spread and uncontrolled use of antimicrobials in both veterinary and human health (Cardinale et al., 2003;Pollett et al., 2012).Recent studies in developed countries have also showed increased resistance to flouroquinolones and macrolides (Gu et al., 2009;Marinou et al., 2012).There is limited information on the prevalence of antimicrobial resistance in poultry in most developing countries, including Nigeria.This study was designed to determine the antimicrobial resistance of thermophilic Campylobacter species isolated from chickens in Nigeria.

MATERIALS AND METHODS
A total of 40 Campylobacter isolates (C.jejuni = 24; C. coli = 16) from a collection of 68 strains isolated from the caecal samples of chickens submitted for routine diagnostic tests to a central Diagnostic laboratory located in Plateau state, north central Nigeria, from different parts of the country between 2008 -2009 was used for this study.The remaining 28 isolates could not be recovered on culture.The caecal contents were cultured on modified charcoal, cefoperazone deoxycholate agar (MCCDA), (Oxoid, Basingstoke, UK) and incubated at 42 o C under microaerobic condition generated by CampyGen R (Oxoid) for 48 hr.The identification of Campylobacter species was based on colony and microscopic morphology, motility, oxidase, catalase, hippurate hydrolysis and indoxyl acetate tests.The isolates were confirmed using multiplex PCR as described by Wang et al. (2002).Susceptibility testing was performed by a broth micro dilution method with MICRONAUT -S anaerob test plates (Merlin Diagnostika, GmbH, Germany) following the Clinical and Laboratory Standards Institute (CLSI, formally NCCLS) guidelines (CLSI, 2007) as described by the manufacturer.We tested the following ten (10) antimicrobial agents at the indicated concentration ranges: ciprofloxacin and azithromycin 0.0625 -8 µg/ml; clindamycin, tetracycline and gentamicin 0.125 -16 µg/ml; erythromycin, streptomycin and chloramphenicol 0.5 -64 µg/ml; nalidixic acid 1 -128 µg/ml and trimethoprim/sulphamethoxazole 0.0625/1.1875-8/152 µg/ml.Isolates were grown on Mueller -Hinton agar plates (Oxoid) with 5% horse blood and were incubated for 48 h at 42C in a microaerobic atmosphere (10 % CO2, 5 % O2 and 85 % N2).Several colonies of Campylobacter were transferred into 2 ml Wilkins Chalgren broth (Sigma -Aldrich) until the turbidity matches a McFarland of 0.5.Then 200 µl of the bacteria suspension was pipetted into 11 ml Mueller -Hinton II broth supplemented with 2.5 % laked horse blood and homogenized well.One hundred microliter (100 µl) of the suspension was inoculated into each well of the 96 well MICRONAUT -S plate using a multichannel pipette.After the inoculation the plates were covered with the perforated plate sealer and incubated at 42C for 24 -48 h under microaerobic conditions (10 % CO2, 5 % O2 and 85 % N2).After incubation, the plates were removed and read visually under a black background.The MICs were defined as the lowest concentration where no viability was observed in the wells of the microplates after incubation.

Statistical analysis
The frequencies of resistance were tested between Campylobacter species using the Fisher's Exact Test for R x C contingency table (R = Rows and C = columns) using Microsoft Excel software for Windows.

DISCUSSION
Results from recent susceptibility studies of Campylobacter species from poultry and poultry meat performed in different countries indicate substantial variation between countries.High resistance rates have been reported from Belgium (Habib et al., 2009), USA (Ge et al., 2003), Italy (Pezzotti et al., 2003;Nobile et al., 2013;Giacomelli et al., 2014) and Czech Republic (Bardon et al., 2008) while lower resistance rates have been reported from Australia (Miflin et al., 2007).Possible explanation for these differences has been due to different National and Regional policies in relation to the use of antimicrobial agents for food animals (Anderson et al., 2006).In this study, there was a high resistance to quinolones (ciprofloxacin and nalidixic acid) among the isolates.High resistance to flouroquinolones in poultry and poultry meat have been reported in many European countries, 72% in the Czech Republic (Bardon et al., 2008), 65% in Turkey (Cokal et al., 2008), and 82% in Spain (Prats et al., 2000).Similarly, high resistance to ciprofloxacin and nalidixic acid has been reported in other countries, 88 and 91% in Korea (Kang et al., 2006), 69.4 and 75% in Iran (Taremi et al., 2006).In contrast, Norstrom et al. (2007) reported no resistance to quinolones in C. jejuni isolated from broilers in Sweden.
In this study, comparable resistance rates were observed for azithromycin, tetracycline, erythromycin, trimethoprim/sulphamethoxazole and streptomycin.Only 20% of the Campylobacter strains tested were susceptible to the antimicrobial agents tested, this finding provides evidence of potential role of chickens in the circulation of resistant Campylobacter strains in human and thus demands more careful attention on antimicrobial use in poultry production and veterinary medicine.Eighty percent of the strains were resistant to one or more of the antimicrobial agents with 32.5% being multiresistant (defined as resistance to 3 or more antimicrobial agents).This indicates that resistance to these antimicrobial agents in this study further confirm the global emergence of antimicrobial resistance of Campylobacter species.
The predominant multiresistant pattern of ciprofloxacin/nalidixic acid/tetracycline in this study is similar to other studies reported from other countries (Hakanen et al., 2003;Cokal et al., 2008;Nobile et al., 2013).It was observed that among isolates that were multi-resistant, ciprofloxacin, nalidixic acid, tetracycline and trimethoprim/sulphamethoxazole were prevalent in most patterns.This is in agreement with the findings of Rodrigo et al. (2007).Resistance of Campylobacter strains in our study demonstrates a high resistance to ciprofloxacin, nalidixic acid, tetracycline and trimetoprim/sulphamethoxazole with higher resistance frequency in the C. jejuni than the C. coli strains.In a recent study by Lemos et al. (2015), a high resistance to nalidixic acid (100%), norfloxacin (100%), ciprofloxacin (95.8%), ampicillin (91.6%) and tetracycline (75%) was observed among Campylobacter species isolated from the liver of chickens.The high resistance profile reported in this study could be as a result of indiscriminate use of these drugs in poultry feed or treatment with flouroquinolones, macrolide and tetracycline in cases of gastroenteritis in chicks which is common in Nigeria.Tet (O) and mutation in the gyrA gene to Thr -86 -Ile have been reported to be responsible for resistance to tetracycline and flouroquinolones repectively (Ge et al., 2003;Ekkapobyotin et al., 2008).In Nigeria, as in other developing countries, although regulations exist on the use of antimicrobial agents, their enforcement is always a problem and virtually non-existent.Though the sample size is relatively small, it gives a fair picture of the situation in the study area.Lower resistance was observed to azithromycin, erythromycin, gentamicin and streptomycin; this may be because these drugs are not commonly used to treat poultry diseases in Nigeria.All the strains were susceptible to clindamycin and chloramphenicol, thus, may be drugs of choice for the treatment of campylobacteriosis.
The occurrence of resistance to ciprofloxacin and multiresistant isolates in this study is of major concern since Campylobacter is a gramnegative organism which could transfer resistant genes to other gramnegative pathogens in the environment (Barlow et al., 2004).This could also have therapeutic implications in the treatment of human bacterial diseases originating from consuming contaminated poultry meat.This study demonstrates that Diagnostic laboratories could be used as surveillance points for antimicrobial resistance for Campylobacter species and other bacteria.Further investigation and surveillance is necessary, especially in developing countries to determine the extent of the resistant situation and proper control measures.

Table 1 .
Antimicrobial resistance of Campylobacter isolates from chickens.

Table 2 .
Multi-resistance patterns among isolates of Campylobacter species sulphamethoxazole (22.5%) and streptomycin (17%).A significantly higher resistance to ciprofloxacin, nalidixic acid and tetracycline was recorded (P < 0.05).Considering the resistance by species, C. jejuni were significantly more resistant than C. coli to ciprofloxacin, nalidixic acid and trimethoprim/sulphamethoxazole (P < 0.05).On the other hand, C. coli were significantly more resistant than C. jejuni to erythromycin